Streptococcus pyogenes (Group A streptococcus) is a Gram-positive, nonmotile, non-sporeforming bacterium that occurs in chains or in pairs of cells, where individual cells are round-to-ovoid cocci, 0.6-1.0 micrometer in diameter. The cell surface structure of Group A streptococci is composed of repeating units of N-acetylglucosamine and N-acetylmuramic acid, the standard peptidoglycan. Historically, the definitive identification of streptococci has rested on the serologic reactivity of “cell wall” polysaccharide antigens as originally described by Rebecca Lancefield. Eighteen group-specific antigens (Lancefield groups) were established. The Group A capsular polysaccharide (also called “C substance” or “group carbohydrate antigen”) is a polymer of N-acetylglucosamine and rhamnose. Some group antigens are shared by more than one species. (K. Todar, Online Textbook of Bacteriology; See textbookofbacteriology.net).
S. pyogenes is one of the most frequent pathogens of humans. Approximately 5-15% of normal individuals harbor the bacterium, usually in the respiratory tract, yet remain asymptomatic. As normal flora, S. pyogenes can infect when defenses are compromised or when the organisms are able to penetrate the constitutive defenses. When the bacteria are introduced or transmitted to vulnerable tissues, a variety of types of suppurative infections can occur.
Acute diseases associated with S. pyogenes occur mainly in respiratory tract, bloodstream or skin. Streptococcal disease is most often a respiratory infection (pharyngitis or tonsillitis) or a skin infection (pyoderma). Acute S. pyogenes infections may present as pharyngitis (strep throat), scarlet fever (rash), impetigo (infection of the superficial layers of the skin) or cellulitis (infection of the deep layers of the skin). Invasive, toxigenic infections can result in necrotizing fasciitis, joint or bone infections, myositis, meningitis, endocarditis and streptococcal toxic shock syndrome. Patients may also develop immune-mediated post-streptococcal sequelae, such as acute rheumatic fever and acute glomerulonephritis, following acute infections caused by S. pyogenes, which occur in 1-3% of untreated infections. These conditions and their pathology are not attributable to dissemination of bacteria, but to aberrant immunological reactions to Group A streptococcal antigens.
Because penicillin is effective in treatment of Group A streptococcal disease, the majority of infections amount to no more than pharyngitis accompanied by a rash. However, due to the occasional cases of rapidly progressive disease and because of the small risk of serious sequelae in untreated infections, S. pyogenes remains a major health concern, and effort is being directed toward clarifying the risk and mechanisms of these sequelae and identifying rheumatogenic and nephritogenic strains of streptococci.
The cell surface of S. pyogenes accounts for many of the bacterium's determinants of virulence, especially those concerned with colonization and evasion of phagocytosis and the host immune responses. The surface of the bacterium is incredibly complex and chemically-diverse. Antigenic components include capsular polysaccharide (C-substance), cell wall peptidoglycan and lipoteichoic acid (LTA), and a variety of surface proteins, including M protein, fimbrial proteins, fibronectin-binding proteins, (e.g. Protein F) and cell-bound streptokinase.
The cytoplasmic membrane of S. pyogenes contains some antigens similar to those of human cardiac, skeletal, and smooth muscle, heart valve fibroblasts, and neuronal tissues. Molecular mimicry between pathogen and host has been proposed as a mechanism for the development of autoimmune diseases. Because microorganisms contain proteins similar to host proteins, the host's immune response may be suppressed or tolerant to infection. Conversely, stimulation of the host's B and T cells by a molecular mimic can cause the host's immune system to begin responding to self proteins as if they are foreign.
As in other autoimmune diseases, both environmental and genetic factors are involved in the development of rheumatic carditis and inflammatory heart disease, and molecular mimicry between the group A streptococcus and heart tissues appears to play a role. The study of B and T cell responses against group A streptococcal antigens has yielded some information about several steps in the pathogenesis of rheumatic carditis following group A streptococcal infection. An early step involves the development of crossreactive autoantibodies against the group A streptococcal carbohydrate antigen N-acetyl-glucosamine and cardiac myosin. These antibodies then react with valvular endothelium, which becomes inflamed with expression of vascular cell adhesion molecule-1 (VCAM-1). T cells, CD4+ and CD8+, then infiltrate through the endothelium/endocardium into the valve (an avascular structure). Aschoff bodies or granulomatous lesions may form containing macrophages and T cells underneath the endocardium. The T cells are responsive to streptococcal M protein antigen sequences. The valve becomes scarred with eventual neovascularization and progressive, chronic disease in the valve. In the host, the mimicking antigens cardiac myosin and laminin have been involved in the myocardium and valve, respectively. (Cunningham, Front. Biosci., 2003, 8:s533-43).
Rheumatic fever (RF) and the antiphospholipid syndrome (APS) are autoimmune diseases sharing similar cardiac and neurological pathologies. There appears to be a considerable overlap of humoral immunity in RF and APS, supporting a hypothesis that common pathogenic mechanisms underlie the development of cardiac valve lesions and Central Nervous System abnormalities in both diseases. The pathogenic molecules engaged in these autoimmune conditions, M protein, N-acetyl-beta-D-glucosamine (also called “NAG” or “GlcNAc”) and beta2 glycoprotein-I (beta2GPI), were found to share some epitopes. The immunoglobulin G sera from APS patients contained a considerable anti-streptococcal M protein as well as anti-GlcNAc activity. Furthermore, beta2GPI inhibited anti-GlcNAc activity from APS patients with chorea. (Blank, et al., 2006, Rheumatology (Oxford). 45(7):833-41).
Detection of microbial pathogens in biological samples is of particular value in clinical medicine, as treatment may vary considerably depending upon the causative organism. Thus, the accurate and rapid identification of pathogens in biological samples of patients suspected of having an infectious disease can be critical to provide prompt and appropriate treatment to patients. Rapid identification of disease-causing organisms in biological samples is important even for non-life threatening infections.
Rapid methods of diagnosing microbial infections have been developed to provide timely results for guiding clinical therapy. Some of the most effective of these rapid methods have been immunologically based. Monoclonal and polyclonal antibodies to microbe-specific antigens have been developed and used in immunoassays to identify specific microbes in biological samples. For example, immunoassays for the identification of group A streptococcal antigens in human samples are useful for the early detection of S. pyogenes infection, so that proper antibiotic treatment may be started.
Group A Streptococcus in pharyngeal exudates can be identified by polyclonal antibodies to antigens specific for Group A streptococcus. One such test is described in U.S. Pat. No. 5,770,460, providing a one-step lateral flow assay for Group A streptococcus-specific antigens. However, tests relying on pharyngeal swabs are often complicated by a high false positive rate. Although instructions for use of pharyngeal swab tests specifically direct the user to avoid contacting the tongue, cheek and/or teeth with the swab, inadvertent contact often occurs, nonetheless. Epithelial cells originating from the tongue, cheek and/or teeth may contain molecular mimics of one or more components of the S. pyogenes cell wall, and the polyclonal antibody specific for Group A streptococcus may bind and “recognize” epitopes on the epithelial cells in a test subject not infected by or carrying Group A strep, resulting in a false positive result. A highly specific and facile immunoassay with a reduced rate of false positives is needed to provide accurate detection of Group A streptococcus infection. Quite surprisingly, the present disclosure fulfills these and other related needs.